Petunia peroxidase a: isolation, purification and characteristics

The fast‐moving anionic peroxidase isoenzyme variant PRXa was purified from leaves of petunia ( Petunia hybrida ). Over 1300‐fold purification was achieved by subjecting extracellular extracts to two sequential acetone precipitations and resuspending the pellets at pH 5.0 and pH 8.0, respectively, followed by gel filtration and chromatofocusing. The purified enzyme had an absorbance ratio ( A 405 nm / A 280 nm ) of 3.6, a molecular mass of about 37 kDa and a pI of 3.8. Three molecular forms with slightly different molecular masses were separated by concanavalin‐A–Sepharose affinity chromatography, indicating that these three forms differ in their carbohydrate moieties. The absorption spectrum of PRXa had maxima at 496 and 636 nm and a Soret band at 405 nm. Spectra of compounds I and IV were obtained by titrating a batch of PRXa stored for several months at ‐20°C with H 2 O 2 . The addition of 1 mol H 2 O 2 /mol freshly purified PRXa caused the formation of compound II, indicating that freshly isolated PRXa contains a bound hydrogen donor which is lost upon storage. Compound III was obtained from both preparations in the presence of excess H 2 O 2 . The pH optimum of PRXa for the reaction with H 2 O 2 and guaiacol was 5.0 and its specific activity 61 mkat/g protein. Among various aromatic compounds, coniferyl alcohol was polymerized by PRXa to presumed lignin‐like material. The extracellular localization and high affinity of PRXa for the cinnamic acid derivatives suggest that this isoenzyme functions in the polymerization or cross‐linking of lignin in the plant cell wall.